Design, synthesis, and characterization of new embelin derivatives as potent inhibitors of X-linked inhibitor of apoptosis protein

Article abstract of DOI:10.1016/j.bmcl.2006.08.072

X-Linked inhibitor of apoptosis protein (XIAP) is a promising molecular target for the design of new anticancer drugs aiming at promoting apoptosis in cancer cells. We have previously identified embelin as an inhibitor of XIAP through computational struct

Full text of DOI:10.1016/j.bmcl.2006.08.072

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5805–5808
Design, synthesis, and characterization of new embelin derivatives
as potent inhibitors of X-linked inhibitor of apoptosis protein
Jianyong Chen, Zaneta Nikolovska-Coleska,
Guoping Wang, Su Qiu and Shaomeng Wang*
Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology and Medicinal Chemistry,
University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA
Received 24 July 2006; revised 15 August 2006; accepted 15 August 2006
Available online 8 September 2006
Abstract—X-Linked inhibitor of apoptosis protein (XIAP) is a promising molecular target for the design of new anticancer drugs
aiming at promoting apoptosis in cancer cells. We have previously identified embelin as an inhibitor of XIAP through computation-
al structure-based database screening. Herein, we report the design, synthesis, and evaluation of new embelin analogues as inhibitors
of XIAP. Our efforts led to the identification of new and more potent inhibitors. For example, compound 6g has a Ki value of
180 nM binding to XIAP BIR3, in a competitive binding assay and represents a promising lead compound for further optimization.
Ó 2006 Elsevier Ltd. All rights reserved.
Apoptosis, or programmed cell death, is a genetically
regulated cell death mechanism.1 Dysfunction of the
apoptosis machinery plays a major role in many human
diseases, including cancer. X-Linked inhibitor of apop-
tosis protein (XIAP) is a potent and effective cellular
inhibitor of apoptosis.2–5 XIAP has been found to be
overexpressed in many human cancer cell lines.6 XIAP
is considered as a promising cancer therapeutic target
because inhibition of XIAP can promote apoptosis in
cancer cells with overexpression of XIAP and sensitize
cancer cells to apoptosis induction.7 One of the major
cellular functions of XIAP is the inhibition of the activ-
ity of caspase-9 by binding to caspase-9 through its
BIR3 domain and trapping caspase-9 in its inactive
form.8 Smac/DIABLO protein (second mitochondria-
derived activator of caspase or direct IAP-binding pro-
tein with low pI) has been discovered as an endogenous
cellular inhibitor of XIAP,9,10 and promotes apoptosis
in cells at least in part by binding to the BIR3 domain
of XIAP where caspase-9 binds and relieving the inhibi-
tion of XIAP to caspase-9.11 There is a strong research
interest in the design of peptidomimetics and non-pep-
tidic small-molecule inhibitors to target the XIAP
BIR3 domain where Smac and caspase-9 bind.12–20 Such
small-molecule inhibitors are predicted to promote
apoptosis in cancer cells and may have great therapeutic
potential to be developed as an entirely new class of
anticancer drugs.
OH
O
-
O
CO2
H3+N
N
N
H
N
O
O
O
OH
1 (Embelin)
H
Smac AVPI Peptide
Through structure-based database searching, we have
previously discovered embelin as a fairly potent, non-
peptide, small-molecule inhibitor of XIAP.20 Embelin
was determined to bind to the XIAP BIR3 domain with
a Ki value of 0.40 lM in our competitive fluorescence-
polarization (FP)-based assay (Table 1). To the best of
our knowledge, embelin is the only known class of
non-peptide inhibitor that binds to the XIAP BIR3
domain, whose chemical structure is not related to the
AVPI peptide in Smac. Hence, embelin represents a
promising initial lead for optimization toward our ulti-
mate goal of developing a new class of anticancer drugs
to target XIAP. In this paper, we wish to report our de-
sign, synthesis, and biochemical evaluation of a series of
new embelin analogues as inhibitors of XIAP.
Keywords: XIAP; Embelin; Small-molecule inhibitors.
*
Corresponding author. Tel.: +1 734 615 0362; fax: +1 734 647
9647; e-mail: shaomeng@umich.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.072

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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5805–5808
Table 1. Binding affinities to the XIAP BIR3 in an FP-based binding
assay for 6a–g
lished quantitative fluorescence-polarization (FP)-based
competitive binding assay24 and compared directly to
embelin (1) and the AVPI Smac peptide. The results
are summarized in Table 1. In our FP-based binding as-
say, embelin (1) and the Smac AVPI peptide were deter-
mined to have Ki values of 0.40 and 0.58 lM,
respectively.
OH
O
O
R
OH
Compound 6a was designed to test the importance of
the C11H23 long hydrophobic tail, in which the C11H23
tail was replaced by a much shorter ethyl group. Our
FP-based binding assay showed that compound 6a has
a Ki value of 10.4 lM to XIAP BIR3, thus 25 times less
potent than embelin. This suggests that the C11H23 long
hydrophobic tail is critical for the binding of embelin to
XIAP BIR3. Based upon this result, we have designed
and synthesized compound 6b with an n-octyl side
chain. Compound 6b has a Ki value of 1.25 lM. Hence,
although compound 6b is 3 times less potent than embe-
lin, it is 8 times more potent than compound 6a, further
confirming the importance of the long hydrophobic tail
in the binding of embelin to XIAP BIR3.
Compound
R
Ki SD (lM)
FP-based binding
assay
1
0.40 0.13
10.4 1.3
1.25 0.9
1.3 0.3
6a
6b
6c
6d
6e
6f
6g
H
–CH2CH2CH2CH2CH2Me
-CH2CH2
The crystal and NMR structures of XIAP BIR3 in com-
plex with Smac protein or peptide showed that the bind-
ing of Smac to XIAP BIR3 is mediated primarily by the
AVPI four amino acid residues in Smac and a well-de-
fined surface binding groove in XIAP BIR3.25,26 While
the alanine residue in the Smac AVPI-binding motif
forms an extensive hydrogen bonding network with
XIAP BIR3, the proline residue has hydrophobic con-
tacts with Trp323 in XIAP. Our modeling suggested that
the hydrophilic dihydroxyquinone core in embelin mim-
ics the alanine residue in the Smac AVPI peptide to form
a hydrogen bonding network. We have designed two
new analogues, 6c and 6d, to explore whether a phenyl
ring would be able to mimic the interaction between
the proline ring in the Smac AVPI peptide and Trp323
in XIAP. As can be seen, while compound 6c with a
(CH2)4 linker between the dihydroxyquinone group
and the phenyl ring has a Ki value of 1.3 lM, compound
6d with a (CH2)2 linker has a Ki value of 0.71 lM.
Hence, compound 6d is 2 and 15 times more potent than
6c and 6a, respectively. Thus, we have made further
modifications based upon compound 6d.
0.71 0.17
0.48 0.3
0.38 0.09
0.18 0.09
Embelin consists of the dihydroxyquinone core and a
long hydrophobic tail. Our modeling predicted that
the hydrophilic dihydroxyquinone core forms a number
of hydrogen bonds with XIAP and the hydrophobic tail
interacts with the hydrophobic pocket where the isoleu-
cine residues in the AVPI Smac peptide bind. In the
present study, we have kept the dihydroxyquinone core
intact and focused our modifications on the hydropho-
bic tail portion of the molecule.
The isoleucine residue in the Smac AVPI peptide binds
to a hydrophobic pocket in XIAP BIR3 and plays an
important role for their binding. Our modeling suggest-
ed that the long hydrophobic tail in embelin interacts
with this hydrophobic pocket in XIAP. We have thus
designed and synthesized a series of new analogues
based upon compound 6d in an attempt to capture the
hydrophobic interaction between the isoleucine residue
in the Smac AVPI peptide and the hydrophobic pocket
in XIAP BIR3.
A series of embelin analogues 6a–g with different hydro-
phobic tails were designed and synthesized. The synthe-
sis of compounds 6a–g is shown in Scheme 1. Briefly,
commercially available or easily prepared corresponding
triphenylphosphonium bromide 2a–g were treated with
1:1 equivalent of n-butyllithium, followed by reaction
with aldehyde 3, which was prepared according to a
published method21 and hydrogenation afforded the
key intermediates 4a–g. Oxidation of 4a–g with ceric
ammonium nitrate gave [1,2]benzoquinones 5a–g.22
The final target compounds 6a–g were obtained by the
treatment of 5a–g with 70% perchloric acid and concen-
trated hydrochloric acid at room temperature for 48 h.23
Compound 6e with an n-butyl group attached to the
meta-position on the phenyl ring in compound 6d has
a Ki value of 0.48 lM, which is slightly more potent than
6d. Encouraged by this result, we replaced the n-butyl
group with a phenylethyl group since it was previously
shown that replacement of the isoleucine in the Smac
Compounds 6a–g were tested for their binding affinities
to recombinant XIAP BIR3 protein using our estab-

J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5805–5808
OMe
5807
OH
O
OMe
OMe
O
O
OMe
c
a,b
d
RCH2PPh3Br
+
R
R
O
R
O
O
CHO
O
O
O
OH
O
2a-g
3
4a-g
5a-g
6a-g
Scheme 1. Synthesis of compounds 6a–g. Reagents and conditions: (a) n-BuLi, THF, 0 °C, 10 min; (b) H2, 10% Pd–C, EtOAc, room temperature;
(c) CAN, CH3CN–H2O, 0 °C, 1 h; (d) HClO4, HCl, dioxane, room temperature, 48 h.
AVPI peptide by a phenylalanine residue increased the
binding affinity of the resulting peptide.27 This resulted
in compound 6f, which has a Ki value of 0.38 lM bind-
ing to XIAP and is as potent as embelin.
ultimate goal of developing a new class of anticancer
drugs by targeting XIAP and promoting apoptosis in
cancer cells.
Compound 6g was designed and synthesized to further ex-
plore the interaction between the terminal phenyl ring and
XIAP by installation of a methyl group on the meta-posi-
tion the binding affinity of the resulting peptide.28 Com-
pound 6g has a Ki value of 0.18 lM and is thus 2 times
more potent than embelin for binding to XIAP BIR3.
Acknowledgments
We are grateful for the financial support from the
Department of Defense Prostate Cancer Program
(PC030410), the Susan G. Komen Foundation, and
the National Cancer Institute, National Institutes of
Health (R21CA104802).
We have evaluated compound 6g for its activity in inhi-
bition of cell growth in the MDA-MB-231 (2LMP) hu-
man breast cancer line and the PC-3 human prostate
cancer cell line. Both of these two cancer cell lines have
high levels of XIAP (data not shown). The results are
shown in Figure 1. As can be seen, compound 6g is effec-
tive in inhibition of cell growth with IC50 values of 5.0
and 5.5 lM in the MDA-MB-231 and PC-3 cell lines,
respectively.
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